1. Field of the Invention
The present invention relates generally to the field of medical microbiology, immunology, vaccines and the prevention of infection by a bacterial pathogen by immunization.
2. Description of the Related Art
Meningococcal meningitis is an infection of the meninges, the thin lining that surrounds the brain and the spinal cord. The causative agent, Neisseria meningitidis (the meningococcus), was identified in 1887. Meningococcal disease was first reported in 1805 when an outbreak swept through Geneva, Switzerland.
Twelve subtypes or serogroups of N. meningitidis have been identified and four (N. meningitidis. A, B, C and W-135) are known to cause epidemics. The pathogenicity, immunogenicity, and epidemic capabilities differ according to the serogroup. Thus the identification of the serogroup responsible for a sporadic case is crucial for epidemic containment. The most common symptoms are stiff neck, high fever, sensitivity to light, confusion, headaches, and vomiting. Even when the disease is diagnosed early and adequate therapy instituted, 5% to 10% of patients die, typically within 24-48 hours of the onset of symptoms. Bacterial meningitis may result in brain damage, hearing loss, or learning disability in 10 to 20% of survivors. A less common but more severe (often fatal) form of meningococcal disease is meningococcal septicaemia which is characterized by a haemorrhagic rash and rapid circulatory collapse.
Major African epidemics are associated with N. meningitidis serogroups A, W-135 and C, and serogroup A is usually the cause of meningococcal disease in Asia. Outside Africa, only Mongolia reported a large epidemic in recent years (1994-95). There is increasing evidence of serogroup W-135 being associated with outbreaks of considerable size. In 2000 and 2001 several hundred pilgrims attending the Hajj in Saudi Arabia were infected with N. meningitidis W-135. Then in 2002, W-135 emerged in Burkina Faso, striking 13,000 people and killing 1,500.
The highest burden of meningococcal disease occurs in sub-Saharan Africa, which is known as the “Meningitis Belt”, an area that stretches from Senegal in the west to Ethiopia in the east, with an estimated total population of 300 million people. This hyper-endemic area is characterized by particular climate and social habits. During the dry season, between December and June, because of dust winds and upper respiratory tract infections due to cold nights, the local immunity is diminished, increasing the risk of meningitis. At the same time, the transmission of N. meningitidis is favored by overcrowded housing at the family level and by large population displacements due to pilgrimages and traditional markets at the regional level. This conjunction of factors explains the large epidemics which occur during this season in the meningitis belt area. Due to herd immunity (whereby transmission is blocked when a critical percentage of the population has been vaccinated, thus extending protection to the unvaccinated), these epidemics occur in a cyclic mode. N. meningitidis A, C and W-135 are now the main serogroups involved in the meningococcal meningitis activity in Africa.
In 1996, Africa experienced the largest recorded outbreak of epidemic meningitis in history, with over 250,000 cases and 25,000 deaths registered. Between that crisis and 2002, 223,000 new cases of meningococcal meningitis were reported to the World Health Organization. The countries most affected have been Burkina Faso, Chad, Ethiopia, and Niger. In 2002, the outbreaks occurring in Burkina Faso, Ethiopia, and Niger accounted for about 65% of the total cases reported on the African continent. Furthermore, the meningitis belt appears to be extending further south. In 2002, the Great Lakes region was affected by outbreaks in villages and refugee camps which caused more than 2,200 cases, including 200 deaths.
In 2006 and 2007, outbreaks of the disease occurred in the North of Ivory Coast and the southern region of Burkina Faso, Southern Sudan and UgaNAD (Nicotinamide adenine dinucleotide), killing several children and adults. Meningococcal meningitis is not only important in Africa but also throughout the world. Meningococcal meningitis is considered an important disease not only for sub-Saharan Africa but also for North America, UK, Ireland, Europe, South East Asia, the Middle East, and New Zealand.
The capsular polysaccharides of Neisseria meningitidis are attractive vaccine candidates because they constitute the most highly conserved and most exposed bacterial-surface antigens. The use of capsular polysaccharides as immunoprophylactic agents against human disease caused by encapsulated bacteria is now firmly established. The capsular polysaccharides of the meningococcus are negatively charged and are obtained in a high molecular-weight immunogenic form by precipitation. Meningococcal polysaccharide vaccines are efficacious for protection from meningitis disease in adults. The duration of protection elicited by the meningococcal polysaccharide vaccines is not long lasting, and has been estimated to be 18 months in adults and children above four years of age. For children from one to four years old the duration of protection is less than three years.
Polysaccharides themselves are poor at stimulating an effective antibody response in the highest risk age groups (infants). Coupling T-cell independent saccharides to a T-cell dependent protein allows the infant immune system to provide T-cell help to B-cells to produce a boostable IgG antibody of high affinity to the polysaccharide antigen. T-Independent antigens are immunologically important. Molecules such as polysaccharides that have numerous identical evenly spaced epitopes characterize one type of TI antigen. As clusters of B-cell receptors bind the antigen simultaneously, it causes B-cell activation without the help of T-helper cells. These antigens are particularly important in young children who respond poorly to these antigens. Children less than two years of age are more susceptible to diseases caused by microbes that have polysaccharide capsules such as Neisseria meningitidis. Discovery of low-cost manufacture of meningitis vaccine is the real objective of this invention to provide affordable vaccine to third world countries to reduce mortality of infants, children, and adults.
Existing State of the Art:
The capsular polysaccharides of Neisseria meningitidis are attractive vaccine candidates because they constitute the most highly conserved and most exposed bacterial-surface antigens (Jennings 1990. Microbial. Immunol. 150, 97-127).
The use of capsular polysaccharides as immunoprophylactic agents against human disease caused by encapsulated bacteria is now firmly established. The capsular polysaccharides of the meningococcus are negatively charged and are obtained in a high molecular weight immunogenic form by precipitation. Meningococcal polysaccharide vaccines are efficacious to protect from meningitis disease in adults (Artenstein, M. S., et al., (1970) New Engl. J. Med. 282, pp. 417-420 and Peltola, H., et al., (1997) New Engl. J. Med 297, pp. 686-691), but cannot provide full protection to infants under the age of 5 (Reingold, A. L., et al., (1985) Lancet 2, pp. 114-118).
The duration of protection elicited by the meningococcal polysaccharide vaccines is not long lasting in adults and children above four years of age (Brandt, B. L. and Artenstein, M. S. (1975) J. Infect. Diseases. 131, pp. S69-S72, Kyhty, H., et al., (1980) J. Infect. Diseases. 142, pp. 861-868, and Cessey, S. J., et al., (1993) J. Infect. Diseases. 167, pp 1212-1216).
For children from one to four years old the duration of protection is less than three years (Reingold, A. L., et al., (1985) Lancet 2, pp. 114-118).
Protective immunity to encapsulated bacterial pathogens such as N. meningitidis is principally mediated by the reaction between antibody and capsular polysaccharide epitopes. In encapsulated gram-negative bacteria, protection results primarily from a direct complement-mediated bactericidal effect (Nahm, M. H., M. A. Apicella, and D. E. Briles. 1999. Immunity to extracellular bacteria, p. 1373-1386. In W. E. Paul (ed.), FuNAD (Nicotinamide adenine dinucleotide) mental immunology, 4th ed. Lippincott-Raven Publishers, Philadelphia, Pa.).
Vaccines have been prepared from the capsular polysaccharides of Neisseria meningitidis (groups A, C, W-135, and Y). These and other polysaccharides have been classified as T cell-independent type 2 (TI-2) antigens based on their inability to stimulate an immune response in animals that carry an X-linked immune B-cell defect (xid) (Mond, J. J., A. Lees, and C. M. Snapper. 1995. T cell-independent antigens type 2. Annu. Rev. Immunol. 13:655-692).
TI-2 antigens tend to be characterized by high molecular weight, multiple repeat epitopes, slow degradation in vivo, and a failure to stimulate major histocompatibility complex (MHC) type II-mediated T-cell help (Mond, J. J., A. Lees, and C. M. Snapper. 1995. T cell-independent antigens type 2. Annu. Rev. Immunol. 13:655-692 and Dick, W. E., Jr., and M. Beurret. 1989. Glycoconjugates of bacterial carbohydrate antigens. A survey and consideration of design and preparation factors. Contrib. Microbiol. Immunol. 10:48-114).
TI-2 antigens generally are incapable of stimulating an immune response in neonatal humans under 18 months of age. This has spurred attempts to modify the capsular polysaccharides such that vaccines protective for all at-risk groups will result. To date, the most successful approach has been to covalently bind carrier proteins to the polysaccharides, thus engendering a vaccine capable of invoking a T-dependent response (Robbins, J. B., R. Schneerson, P. Anderson, and D. H. Smith. 1996. The 1996 Albert Lasker Medical Research Awards. Prevention of systemic infections, especially meningitis, caused by Haemophilus influenzae type b. Impact on public health and implications for other polysaccharide-based vaccines. JAMA 276:1181-11).
Glucose uptake seems to be affected by oxygen concentration and this effect could be related to different levels of carbohydrate metabolism according to higher or lower availability of oxygen (Fu et al., 1995 Biotechnology., vol. 13, pp. 170-174)
Class 4 proteins of Neisseria meningitidis are known to be anti-bactericidal. A novel methodology for the purification of polysaccharides to produce toxin-free vaccine, where class 4 were deleted from the vaccine strains, was developed (Romero, D and Outschoorn I. M. (1994) Clin. Microb. Rev. 7: 559-575).
Several synthetic media were discovered for large-scale production of meningococcal polysaccharide (Frantz, I. D. Jr. Growth Requirements of the Meningococcus. J. Bact., 43: 757-761, 1942; Catlin, B. W. Nutritional profiles of Neisseria lactamica, gonorrhoeae and meningitidis, in chemically defined media. J. Inf. Dis., 128(2): 178-194, 1973; Watson-Scherp Medium: Watson R G, et al. The specific hapten of group C (group IIa) meningococcus, II. Chemical nature. J Immunol 1958; 81:337-44; Marcelo Fossa da Paz; Júulia Baruque-Ramos; Haroldo Hiss; Márcio Alberto Vicentin; Maria Betania Batista Leal; Isaías Raw. Polysaccharide production in batch process of Neisseria meningitidis serogroup C comparing Frantz, modified Frantz and Catlin 6 cultivation media, Braz. J. Microbiol. vol. 34., no. 1. São Paulo January/April 2003).
Cox et. al., (Andrew D Cox, J Claire Wright, Jianjun Li, Derek W Hood, E Richard Moxon, James C Richards 2003. Phosphorylation of the lipid A region of meningococcal lipopolysaccharide: identification of a family of transferases that add phosphoethanolamine to lipopolysaccharide J Bacteriol. 2003 June; 185 (11):3270-7 12754224.) reported that the NMB1638 gene of Neisseria meningitidis was responsible for a lipopolysaccharide (LPS) containing lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues.
Gotschlich E. C.; Liu, T. Y.; Artenstein, M. D. Human immunity to the meningococcal—III. preparation and immunochemical properties of the group A, group B, and group C meningococcal polysaccharides. J. Exp. Med., 129(2): 1349-1365, 1969 reported effective method for purification of meningococcal polysaccharides from liquid cultures.
Cationic reagent Cetavlon™ (hexadecyltrimethyl ammonium bromide) was used to precipitate anionic polysaccharides in this study as per Aymé, G.; Donikian, R.; Mynard, M. C.; Lagrandeur, G. Production and Controls of Serogroup A Neisseria meningitidisPolysaccharide Vaccine. In: Table Ronde Sur L'Immunoprophilaxie de la Meningite Cerebro-Spinale. Edition Fondation Mérieux, Lyon (France), 1973); Carty, C. E. et al. Cultivation studies of Neisseria meningitidis serogroups A, C, W-135 and Y. Developments in Industrial Microbiology (edited by Merck Laboratories), 25:695-700, 1984.
We have chosen ELISA bioassays for the trials because transportation problems of live bacteria from the United States to Africa for performing SBA bioassays.
Meningococcal serogroup A, C, W-135, and Y polysaccharides and DT or CRM197-based conjugates were prepared as already described (Costantino, P., F. Norelli, A. Giannozzi, S. D'Ascenzi, A. Bartoloni, S. Kaur, D. Tang, R. Seid, S. Viti, R. Paffetti, M. Bigio, C. Pennatini, G. Averani, V. Guarnieri, E. Gallo, N. Ravenscroft, C. Lazzeroni, R. Rappuoli, and C. Ceccarini. 1999. Size fractionation of bacterial capsular polysaccharides for their use in conjugate vaccines. Vaccine 17:1251-1263.; Costantino, P., S. Viti, A. Podda, M. A. Velmonte, L. Nencioni, and R. Rappuoli. 1992. Development and phase 1 clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691-698.; Ravenscroft, N., G. Averani, A. Bartoloni, S. Berti, M. Bigio, V. Carinci, P. Costantino, S. D'Ascenzi, A. Giannozzi, F. Norelli, C. Pennatini, D. Proietti, C. Ceccarini, and P. Cescutti. 1999. Size determination of bacterial capsular oligosaccharides used to prepare conjugate vaccines. Vaccine 17:2802-2816.).
The same conjugation chemistry was used for the preparation of Y constructs (Jennings, H. J., and Lugowski, C. 1981. Immunochemistry of group A, B, and C meningococcal polysaccharide-tetanus toxoid conjugates. J. Immunol. 127, 1011-1018). The polysaccharide content of serogroups C, W-135, and Y conjugates was quantified by sialic acid determination (Svennerholm, L. 1957. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta 24:604-611.).
Serogroup A conjugate was quantified by mannosamine-1-phosphate chromatographic determination (Ricci, S., A. Bardotti, S. D'Ascenzi, and N. Ravenscroft. 2001. Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection. Vaccine 19:1989-1997.).
The protein content was measured by a micro-bicinchoninic acid assay of Lowry et al. (1951). The polysaccharide-to-protein ratio of conjugates ranged between 0.3 and 1.5, similar to that of cross-reacting material DT and CRM-based conjugates (Giannini, G., R. Rappuoli, and G. Ratti. 1984. The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res. 12:4063-4069).
A lymphocyte proliferation assay was performed according to the method described by us in our journal article (Reddy J R, Kwang J, Varthakavi V, Lechtenberg K F, Minocha H C. Semiliki forest virus vector carrying the bovine viral diarrhea virus NS3 (p80) cDNA induced immune responses in mice and expressed BVDV protein in mammalian cells. Comp. Immunol. Microbiol. Infect. Dis. 1999 October; 22(4):231-46).
In addition, antigenic variation (Antigenic Variation of the Class-1 Outer Membrane Protein in Hyperendemic Neisseria meningitidis trains in The Netherlands Aldert Bart et. al., Infection and Immunity, 1999, Vol 67 (8) p.3842-3846) and human complement sensitivity of Neisseria meningitidis is a barrier to rely on SBA bioassays.
Conjugation of bacterial polysaccharides to immunogenic carrier proteins generally results in conjugates that induce strong anti-polysaccharide T-helper-cell dependent immune responses in young infants (Granoff, D. M., and S. L. Harris. 2004. Protective activity of group C anticapsular antibodies elicited in two-year-olds by an investigational quadrivalent Neisseria meningitidis-diphtheria toxoid conjugate vaccine. Pediatr. Infect. Dis. J. 23:490-497).
The existing state of the art described in the U.S. Pat. No. 4,123,520 for precipitating polysaccharides by a phenol extraction method is found to require more steps for removing the phenol contaminants from the pure polysaccharide mixture. The problem with the invention disclosed in this patent is that the phenol contaminants may interfere with the pure polysaccharide production process.
The existing state of the art described in the U.S. Pat. No. 4,182,751 for precipitating polysaccharides by a phenol extraction for removing the lipopolysaccharide endotoxin from the pure polysaccharide mixture. The problem with the invention disclosed in this patent is that the phenol contaminants may interfere with the pure polysaccharide production process.
The existing state of art described in patent no. WO03007985 for precipitating the polysaccharide with cetalvon and depolymerized chemical hydrolysis. The problem with that invention is the chemical process for depolymerization may interfere with the purity in polysaccharide vaccines production.
U.S. Pat. No. 5,494,808 reports a large-scale, high cell density (5 g/L dry cell weight, and an optical density of between about 10-13 at 600 nm) fermentation process for the cultivation of N. meningitidis. The problem with the invention art is that large scale biomass production reduces the production of capsular polysaccharides.
Existing art reported in the U.S. patent publication No. 20060088554 about the depolymerization of polysaccharides and conjugation of polysaccharides with carrier proteins which are activated by chemical means. The problem with this invention is that the chemical residues tend to induce adverse side effects during routine immunization.
U.S. patent publication No: 20050002957 reports depolymerization of polysaccharides by chemical means, which results in producing chemical residues and conjugation of polysaccharides with carrier proteins which are activated chemically, requiring more purification steps. The average size of purified capsular polysaccharides is about 8,000 to 35,000 Daltons, which may not provide efficient immune response in humans.
The existing state of the art described in the patent No WO2005004909 reports, including adjuvant for enhancing immunogenicity against Neisseria meningitidis serogroups A, C, W-135, and Y, which may have adverse side effects during routine immunization.
U.S. Pat. No. 6,933,137 claims the development of ‘animal free meningococcal polysaccharide fermentation medium’, containing soy peptone as a nitrogen source. The problem with this medium is that it requires pH adjustment during the fermentation process. Glucose utilization is higher in this medium, resulting in excessive cellular biomass.
U.S. Pat. No. 6,642,017 relates to methods of modulating capsular polysaccharide production in pneumococci such as Streptococcus pneumoniae. This invention of modulating capsular polysaccharide production is not related to N. meningitidis. 
Therefore there is a need for an invention to eliminate the short-comings identified in the above prior art and to invent a method of producing a meningococcal meningitis vaccine without any chemical impurities or residues to eliminate the disadvantage of the present state of the art for depolymerization and conjugation by chemical means and capsular polysaccharide size. Also, there is a need for a medium that ensures a higher yield of polysaccharides and lower yield of cellular biomass to facilitate the production and purification processes for vaccine production.
Therefore it is an object of the present invention to invent a method of producing meningococcal meningitis vaccine comprising N. meningitidis serotypes A, C, Y and W-135 that have long lasting effect and provide broad spectrum immunity to humans of all age groups.
It is yet another object of the present invention to develop a method wherein trace chemical impurities currently present in the available meningococcal meningitis vaccine are eliminated by a mechanical method, preferably sonication.
Another object of the present invention is to invent a composition of a medium that yields a higher percentage of polysaccharides in comparison to known media employed for producing meningococcal meningitis vaccine.
It is yet another object of the present invention to invent a composition of a medium that yields a lower percentage of cellular biomass in comparison with known media employed for producing meningococcal meningitis vaccine.
It is yet another object of the invention to identify an optimum molecular size of N. meningitidis polysaccharides of serogroups A, C, Y and W-135 that confers broad spectrum immunogenic protection against meningitis.